The importance of the Niemann Pick C-1 Like 1 (NPC1L1) protein in cholesterol absorption by the intestine is well recognized. However, whether NPC1L1 is a membrane bound protein serving as a transporter for cholesterol uptake by enterocytes or an intracellular protein responsible for cholesterol trafficking from the apical plasma membrane to intracellular compartments for lipoprotein assembly remains controversial. The NPC1L1 is also expressed at high levels in human liver, but its expression in rodent liver is minimal. Hence, the importance of hepatic NPC1L1 is still unclear. The goals of this study are (i) to identify the physiological mechanism by which NPC1L1 participates in cholesterol absorption in the intestine, and (ii) to delineate the role of liver NPC1L1 in lipoprotein metabolism and homeostasis. Preliminary Results showed that whereas NPC1LT''mice were defective in cholesterol absorption, no difference in cholesterol transport kinetics was observed between intestinal brush border membrane vesicles obtained from NPC1L1+/+ and NPC1LT''mice. Moreover, fluorescent cholesterol was efficiently taken up from intestinal lumen into absorptive epithelial cells of the intestine in both NPC1L1+/+ and NPC1LT''mice, but not in mice treated with the cholesterol absorption inhibitor ezetimibe. These results provided strong support for the hypothesis that NPC1L1 is not the high affinity membrane transporter but an intracellular cholesterol transport protein. Our data also showed that suppression of NPC1L1 activity in the liver lowered VLDL production and inhibited selective uptake of HDL-cholesterol, suggesting that hepatic NPC1L1 may modulate plasma lipoprotein homeostasis. Specific Aim 1 will identify the functional role of NPC1L1 in intestinal lipid transport by mating NPC1L1*/+ and NPC1LT''mice to ABCG5G8'''mice and then determining the distribution and site of cholesterol accumulation in the intestine to further test the hypothesis that NPC1L1 is an intracellular lipid trafficking protein targeting cholesterol after apical uptake to intracellular compartments for lipoprotein assembly. Specific Aim 2 will use in vitro cell culture model to test the hypothesis that NPC1L1 expression is necessary but not sufficient without expression of cell surface cholesterol transporters such as SR-BI and/or CD36 in promoting cholesterol absorption and intracellular transport. Specific Aim 3 will produce transgenic mice expressing NPC1L1 in the liver, to similar extent as its expression level in human liver, to identify the functional significance of hepatic NPC1L1 in plasma lipid homeostasis, VLDL production, selective uptake of HDL-cholesterol, and reverse cholesterol transport. Taken together, these studies will provide a better understanding on the physiological importance and mechanism of action of NPC1L1, potentially offering alternative targets for therapeutic treatment of hypercholesterolemia and lowering the risk of lipid-induced metabolic disorders.